Compositions and methods using complexes of heat shock protein 90 and antigenic molecules for the treatment and prevention of neoplastic diseases

ABSTRACT

The present invention relates to methods and compositions for eliciting an immune response and the prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases. The methods of the invention comprise administering a composition comprising an effective amount of a complex, in which the complex consists essentially of a heat shock protein (hsp) noncovalently bound to an antigenic molecule. &#34;Antigenic molecule&#34; as used herein refers to the peptides with which the hsps are endogenously associated in vivo as well as exogenous antigens/immunogens (i.e., with which the hsps are not complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof. In a preferred embodiment, the complex is autologous to the individual. The effective amounts of the complex are in the range of 10-600 micrograms for complexes comprising hsp7o, 50-1000 micrograms for hsp9o, and 10-600 micrograms for gp96. The invention also provides a method for measuring tumor rejection in viva in an individual, preferably a human, comprising measuring the generation by the individual of MHC Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor. Methods of purifying hsp7o-peptide complexes are also provided.

This invention was made with government support under grant numbersCA44786 and CA64394 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

This is a division, of application Ser. No. 08/527,391, filed Sep. 13,1995 now U.S. Pat. No. 5,837,251.

1. INTRODUCTION

The present invention relates to methods and compositions for theprevention and treatment of infectious diseases, primary and metastaticneoplastic diseases, including, but not limited to human sarcomas andcarcinomas. In the practice of the prevention and treatment ofinfectious diseases and cancer, compositions of complexes of heatshock/stress proteins (hsps) including, but not limited to, hsp70,hsp90, gp96 alone or in combination with each other, noncovalently boundto antigenic molecules, are used to augment the immune response togenotoxic and nongenotoxic factors, tumors and infectious agents.

2. BACKGROUND OP THE INVENTION

The era of tumor immunology began with experiments by Prehn and Main,who showed that antigens on the methylcholanthrene (MCA)-inducedsarcomas were tumor specific in that transplantation assays could notdetect these antigens in normal tissue of the mice (Prehn, R. T., etal., 1957, J. Natl. Cancer Inst. 18:769-778). This notion was confirmedby further experiments demonstrating that tumor specific resistanceagainst MCA-induced tumors can be elicited in the autochthonous host,that is, the mouse in which the tumor originated (Klein, G., et al.,1960, Cancer Res. 20:1561-1572).

In subsequent studies, tumor specific antigens were also found on tumorsinduced with other chemical or physical carcinogens or on spontaneoustumors (Kripke, M. L., 1974, J. Natl. Cancer Inst. 53:1333-1336; Vaage,J., 1968, Cancer Res. 28:2477-2483; Carswell, E. A., et al., 1970, J.Natl. Cancer Inst. 44:1281-1288). Since these studies used protectiveimmunity against the growth of transplanted tumors as the criterion fortumor specific antigens, these antigens are also commonly referred to as"tumor specific transplantation antigens" or "tumor specific rejectionantigens." Several factors can greatly influence the immunogenicity ofthe tumor induced, including, for example, the specific type ofcarcinogen involved, immunocompetence of the host and latency period(Old, L. J., et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; Bartlett, G.L., 1972, J. Natl. Cancer Inst. 49:493-504).

Most, if not all, carcinogens are mutagens which may cause mutation,leading to the expression of tumor specific antigens (Ames, B. N., 1979,Science 204:587-593; Weisburger, J. H., et al., 1981, Science214:401-407). Some carcinogens are immunosuppressive (Malmgren, R. A.,et al., 1952, Proc. Soc. Exp. Biol. Med. 79:484-488). Experimentalevidence suggests that there is a constant inverse correlation betweenimmunogenicity of a tumor and latency period (time between exposure tocarcinogen and tumor appearance) (Old, L. J., et al., 1962, Ann. N.Y.Acad. Sci. 101:80-106; and Bartlett, G. L., 1972, J. Natl. Cancer Inst.49:493-504). Other studies have revealed the existence of tumor specificantigens that do not lead to rejection, but, nevertheless, canpotentially stimulate specific immune responses (Roitt, I., Brostoff, Jand Male, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps.17.1-17.12).

2.1. Tumor-Specific lmunoqenicities of Heat Shock/Stress Proteins hsp70,hsp90 and gp96

Srivastava et al. demonstrated immune response tomethylcholanthrene-induced sarcomas of inbred mice (1988, Immunol. Today9:78-83). In these studies it was found that the molecules responsiblefor the individually distinct immunogenicity of these tumors wereidentified as cell-surface glycoproteins of 96kDa (gp96) andintracellular proteins of 84 to 86kDa (Srivastava, P. K., et al., 1986,Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich, S. J., et al., 1986,Proc. Natl. Acad. Sci. USA 83:3121-3125. Immunization of mice with gp96or p84/86 isolated from a particular tumor rendered the mice immune tothat particular tumor, but not to antigenically distinct tumors.Isolation and characterization of genes encoding gp96 and p84/86revealed significant homology between them, and showed that gp96 andp84/86 were, respectively, the endoplasmic reticular and cytosoliccounterparts of the same heat shock proteins (Srivastava, P. K., et al.,1988, Immunogenetics 28:205-207; Srivastava, P. K., et al., 1991, Curr.Top. Microbiol. Immunol. 167:109-123). Further, hsp70 was shown toelicit immunity to the tumor from which it was isolated but not toantigenically distinct tumors. However, hsp70 depleted of peptides wasfound to lose its immunogenic activity (Udono, M., and Srivastava, P.K., 1993, J. Exp. Med. 178:1391-1396). These observations suggested thatthe heat shock proteins are not immunogenic per se, but are carriers ofantigenic peptides that elicit specific immunity to cancers (Srivastava,P. K., 1993, Adv. Cancer Res. 62:153-177).

2.2. Pathobiology of Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

Pre-malignant abnormal cell growth is exemplified by hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ,jwithout significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

The neoplastic lesion may evolve clonally and develop an increasingcapacity for invasion, growth, metastasis, and heterogeneity, especiallyunder conditions in which the neoplastic cells escape the host's immunesurveillance (Roitt, I., Brostoff, J and Male, D., 1993, Immunology, 3rded., Mosby, St. Louis, pps. 17.1-17.12).

2.3. Immunotherapy

Four basic cell types whose function has been associated with antitumorcell immunity and the elimination of tumor cells from the body are: i)B-lymphocytes which secrete immunoglobulins into the blood plasma foridentifying and labeling the nonself invader cells; ii) monocytes whichsecrete the complement proteins which are responsible for lysing andprocessing the immunogIobulin-coated target invader cells; iii) naturalkiller lymphocytes having two mechanisms for the destruction of tumorcells-antibody-dependent cellular cytotoxicity and natural killing; andiv) T-lymphocytes possessing antigen-specific receptors and eachT-lymphocyte clone having the capacity to recognize a tumorcell-carrying complementary marker molecules (Schreiber, H., 1989, inFundamental Immunology (ed). W. E. Paul, pp. 923-955).

Several factors can influence the immunogenicity of tumors induced.These factors include dose of carcinogen, immunocompetence of the host,and latency period. Immunocompetence of the host during the period ofcancer induction and development can allow the host to respond toimmunogenic tumor cells. This may prevent the outgrowth of these cellsor select far less immunogenic escape variants that have lost theirrespective rejection antigen. Conversely, immunosuppression or immunedeficiency of the host during carcinogenesis or tumorigenesis may allowgrowth of highly immunogenic tumors (Schreiber, H., 1989, in FundamentalImmunology (ed). W. E. Paul, pp. 923-955).

Three major types of cancer immunotherapy are currently being explored:i) adoptive cellular immunotherapy, ii) in vivo manipulation of patientplasma to remove blocking factors or add tumoricidal factors, and iii)in vivo administration of biological response modifiers (e.g.,interferons (IFN; IFN-alpha and IFN-gamma), interleukins (IL;

IL-2, IL-4 and IL-6), colony-stimulating factors, tumor necrosis factor(TNF), monoclonal antibodies and other immunopotentiating agents, suchas corynebacterium parvum (C. parvum) (Kopp, W. C., et al., 1994, CancerChemotherapy and Biol. Response Modifiers 15:226-286). There is littledoubt that immunotherapy of cancer as it stands is falling short of thehopes invested in it. Although numerous immunotherapeutic approacheshave been tested, few of these procedures have proved to be effective asthe sole or even as an adjunct form of cancer prevention and treatment.

2.3.1. Interleukins (IL-2, IL-4 and IL-6)

IL-2 has significant antitumor activity in a small percentage ofpatients with renal cell carcinoma and melanoma. Investigators continueto search for IL-2 based regimens that will increase the response ratesin IL-2 responsive tumors, but, for the most part, have neither definednew indications nor settled fundamental issues, such as whether doseintensity is important in IL-2 therapy (Kopp, W. C., et al., 1994,Cancer Chemotherapy and Biol. Response Modifiers 15:226-286). Numerousreports have documented IL-2 associated toxicity involving increasednitrate levels and the syndrome of vascular leak and hypotension,analogous to septic shock. In addition, an increased incidence ofnonopportunistic bacterial infections and autoimmune complications arefrequently accompanied by the antitumor response of IL-2 (Kopp, W. C.,et al., 1994, Cancer Chemotherapy and Biol. Response Modifiers15:226-286).

IL-4 and IL-6 are also being tested as antitumor agents either directlyor through immunomodulating mechanisms. Dose-limiting toxicities havebeen observed with both agents in Phase I clinical trials (Gilleece, M.H., et al., 1992, Br. J. Cancer 66:204-210, Weber, J., et al., 1993, J.Clin. Oncol. 11:499-506).

2.3.2. Tumor Necrosis Factor

The toxicity of systemically administered TNF seriously limits its usefor the treatment of cancer. TNF has been most effective when used forregional therapy, in which measures, such as limb isolation forperfusion, are taken to limit the systemic dose and hence the toxicityof TNF. Dose-limiting toxicity of TNF consist of thrombocytopenia,headache, confusion and hypotension (Mittleman, A., et al., 1992, Inv.New Drugs 10:183-190).

2.3.3. Interferons

The activity of IFN-α has been described as being modest in a number ofmalignancies, including renal cell carcinoma, melanoma, hairy cellleukemia low-grade non-Hodgkin's lymphoma, and others. Higher doses ofIFN-α are usually associated with higher response rates in somemalignancies, but also cause more toxicity. In addition, more and morereports indicate that relapses after successful interferon therapycoincide with formation of neutralizing antibodies against interferon(Ouesada, J. R., et al., 1987, J. Interferon Res. 67:678.

2.4. Pharmacokinetic Models for Anticancer Chemotherapeutic andImunotherapeutic Drugs: Extrapolation and Scaling of Animal Data toHumans

The ethical and fiscal constraints which require the use of animalmodels for most toxicology research also impose the acceptance ofcertain fundamental assumptions in order to estimate dose potency inhumans from dose-response data in animals. Interspecies dose-responseequivalence is most frequently estimated as the product of a referencespecies dose and a single scaling ratio based on a physiologicalparameter such as body weight, body surface area, maximum life spanpotential, etc. Most frequently, exposure is expressed as milligrams ofdose administered in proportion to body mass in kilograms (mg kg⁻¹).Body mass is a surrogate for body volume, and therefore, the ratiomilligrams per kilogram is actually concentrations in milligrams perliter (Hirshaut, Y., et al., 1969, Cancer Res. 29:1732-1740). The keyassumptions which accompany this practice and contribute to its failureto accurately estimate equipotent exposure among various species are: i)that the biological systems involved are homogeneous, "well-stirredvolumes" with specific gravity equal to 1.0; ii) that the administeredcompounds are instantly and homogeneously distributed throughout thetotal body mass; and iii) that the response of the biological systems isdirectly proportional only to the initial concentration of the testmaterial in the system. As actual pharmacokinetic conditions depart fromthese assumptions, the utility of initial concentration scaling betweenspecies declines.

Through pharmacokinetics, one can study the time course of a drug andits metabolite levels in different fluids, tissues, and excreta of thebody, and the mathematical relationships required to develop models tointerpret such data. It, therefore, provides the basic informationregarding drug distribution, availability, and the resulting toxicity inthe tissues and hence, specifies the limitation in the drug dosage fordifferent treatment schedules and different routes of drugadministration. The ultimate goal of the pharmacokinetic studies ofanticancer drugs is thus to offer a framework for the design of optimaltherapeutic dosage regimens and treatment schedules for individualpatients.

The currently utilized guidelines for prescription have evolvedgradually without always having a complete and explicit justification.In 1966, Freireich and co-workers proposed the use of surface areaproportions for interspecies extrapolation of the acute toxicity ofanticancer drugs. This procedure has become the method of choice formany risk assessment applications (Freireich, E. J., et al., 1966,Cancer Chemotherapy Rep. 50:219-244). For example, surface area scalingis the basis of the National Cancer Institute's interspeciesextrapolation procedure for anti-cancer drugs (Schein, P. S., et al.,1970, Clin. Pharmacol. Therap. 11:3-40; Goldsmith, M. A., et al., 1975,Cancer Res. 35:1354-1364). In accepting surface area extrapolation, thetenuous basis for initial concentration scaling has been replaced by anempirical approach. The basic formula used for estimating prescriptionof cancer chemotherapy per body surface area (BSA) is BSA=k×kg^(2/3), inwhich k is a constant that differs for each age group and species. Forexample, the k value for adult humans is 11, while for mice it is 9 (SeeQuiring, P., 1955, Surface area determination, in Glasser E. (ed.)Medical Physics I Chicago: Medical Year Book, p. 1490 and Vriesendorp,H. M., 1985, Hematol. (Supplm. 16) 13:57-63). The major attraction ofexpressing cancer chemotherapy per m² BSA appears to be that it offersan easily remembered simplification, i.e., equal doses of drug per m²BSA will produce approximately the same effect in comparing differentspecies and age groups. However, simplicity is not proof and alternativemethods for estimating prescription of anticancer drugs appear to have abetter scientific foundation, with the added potential for a moreeffective use of anticancer agents (Hill, J. A., et al., 1989, HealthPhysics 57:395-401).

The effectiveness of an optimal dose of a drug used in chemotherapyand/or immunotherapy can be altered by various factors, including tumorgrowth kinetics, drug resistance of tumor cells, total-body tumor cellburden, toxic effects of chemotherapy and/or immunotherapy on cells andtissues other than the tumor, and distribution of chemotherapeuticagents and/or immunotherapeutic agents within the tissues of thepatient. The greater the size of the primary tumor, the greater theprobability that a large number of cells (drug resistant and drugsensitive) have metastasized before diagnosis and that the patient willrelapse.

Some metastases arise in certain sites in the body where resistance tochemotherapy is based on the limited tissue distribution ofchemotherapeutic drugs administered in standard doses. Such sites act assanctuaries that shield the cancer cells from drugs that are circulatingin the blood; for example, there are barriers in the brain and teststhat impede drug diffusion from the capillaries into the tissue. Thus,these sites may require special forms of treatment such asimmunotherapy, especially since immunosuppression is characteristic ofseveral types of neoplastic diseases.

3. SUMMARY OF THE INVENTION

The methods of the invention comprise methods of eliciting an immuneresponse in an individual in whom the treatment or prevention of canceris desired by administering a composition comprising an effective amountof a complex in which the complex consists essentially of a heat shockprotein (hsp) noncovalently bound to an antigenic molecule. The amountsof the complex are within ranges of effective dosages, discovered by thepresent inventor to be effective, and which are surprisingly smallerthan those amounts predicted to be effective by extrapolation by priorart methods from dosages used in animal studies. In a preferredembodiment, the complex is autologous to the individual; that is, thecomplex is isolated from the cancer cells of the individual himself(e.g., preferably prepared from tumor biopsies of the patient).Alternatively, the hsp and or the antigenic molecule can be isolatedfrom the individual or from others or by recombinant production methodsusing a cloned hsp originally derived from the individual or fromothers. "Antigenic molecule" as used herein refers to the peptides withwhich the hsps are endogenously associated in vivo (e.g., inprecancerous or cancerous tissue), as well as exogenousantigens/immunogens (i.e., with which the hsps are not complexed invivo) or antigenic/immunogenic fragments and derivatives thereof. Suchexogenous antigens and fragments and derivatives (both peptide andnon-peptide) thereof for use in complexing with hsps, can be selectedfrom among those known in the art, as well as those readily identifiedby standard immunoassays known in the art by detecting the ability tobind antibody or MHC molecules (antigenicity) or generate immuneresponse (immunogenicity).

The invention also provides a method for measuring tumor rejection invivo in an individual, preferably a human, comprising measuring thegeneration by the individual of MHC Class I-restricted CD8⁺ cytotoxic Tlymphocytes specific to the tumor.

The invention provides methods for determining doses for treatinginfectious diseases or for human cancer immunotherapy by evaluating theoptimal dose of complexes of hsps noncovalently bound to antigenicmolecules in experimental tumor models and extrapolating the data. Thepresent invention relates to methods and compositions for prevention andtreatment of primary and metastatic neoplastic diseases.

Specific therapeutic regimens, pharmaceutical compositions, and kits areprovided by the invention. In contrast to the prior art, the therapeuticregimen, and corresponding pharmaceutical compounds of the presentinvention are not based on body weight or surface area of the patient.The present inventor has discovered that a dosage substantiallyequivalent to that seen to be effective in smaller non-human mammals(e.y., mice) is effective for human administration, optionally subjectto a correction factor not exceeding a fifty fold increase, based on therelative lymph node sizes in such mammals and in humans. Pharmaceuticalformulations are provided, based on a newly-discovered extrapolation andscaling of animal dosage to human, comprising compositions of complexesof antigenic molecules and heat shock/stress proteins, including but notlimited to hsp70, hsp90, gp96 either alone or in combination.Specifically, interspecies dose-response equivalence for hspnoncovalently bound to antigenic.molecules for a human dose. isestimated as the product of the therapeutic dosage observed in mice anda single scaling ratio, not exceeding a fifty fold increase.

The present invention encompasses methods for prevention and treatmentof cancer by enhancing the host's immune competence and activity ofimmune effector cells. Furthermore, the invention provides methods forevaluating the efficacy of drugs in enhancing immune responses fortreatment and monitoring the progress of patients participating inclinical trials for the treatment of primary and metastatic neoplasticdiseases.

Immunotherapy using the therapeutic regimens of the invention, byadministering such complexes of heat shock/stress proteins noncovalentlybound to antigenic molecules, can-induce specific immunity to tumorcells, and leads to regression of the tumor mass. Cancers which areresponsive to specific immunotherapy by the heat shock/stress proteinsof the invention include but are not limited to human sarcomas andcarcinomas. In a specific embodiment, hsp-antigenic molecule complexesare allogeneic to the patient; in a preferred embodiment, thehsp-antigenic are autologous to (derived from) the patient to whom theyare administered.

Particular compositions of the invention and their properties aredescribed in the sections and subsections which follow. A preferredcomposition comprises hsp-peptide complexes isolated from the tumorbiopsy of the patient to whom the composition is to be administered.Such a composition which comprises hsp70, hsp90 and/or gp96 demonstratesstrong inhibition of a variety of tumors in mammals. Moreover, thetherapeutic doses that are effective in the corresponding experimentalmodel in rodents as described infra, in Section 6 can be used to inhibitthe in vivo growth of colon and liver cancers in human cancer patientsas described in Section 7, infra. Preferred compositions comprisinghsp70, hsp90 and/or gp96 which preferably exhibit no toxicity whenadministered to human subjects are also described.

In another embodiment, the methods further optionally compriseadministering biological response modifiers, e.g., IFN-α, IFN-γ, IL-2,IL-4, IL-6, TNF, or other cytokine growth factors affecting the immunecells, in combination with the hsp complexes.

In addition to cancer therapy, the complexes of hsps noncovalently boundto antigenic molecules can be utilized for the prevention of a varietyof cancers, e.g., in individuals who are predisposed as a result offamilial history or in individuals with an enhanced risk of cancer dueto environmental factors.

An improved method for purification of hsp70-peptide complexes fromcells is also provided.

The Examples presented in Sections 6 and 7 below, detail the useaccording to the methods of the invention of hsp-peptide complexes incancer immunotherapy in experimental tumor models and in human patientssuffering from advanced colon and liver cancer.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1. Effect of Administration of gp96 derived from UV6138 or UV6139SJcarcinomas on tumor growth measured as average tumor diameter (mm).

Panel A: Lane 1, SDS-PAGE profile of gp96 preparation; Lane 2,Immunoblot of lane 1 with antibody specific for gp96.

Panel B (top): All mice were challenged with UV6138 cells. The firstgroup of mice (open circles) received PBS; the second group of mice(solid circles) received gp96 derived from UV6138 cells; and the thirdgroup of mice received gp96 derived from UV6139SJ cells.

Panel B (bottom): All mice were challenged with UV6139SJ cells. Thefirst group of mice (open circles) received PBS; the second group ofmice (solid circles) received gp96 derived from UV6138 cells; and thethird group of mice as received gp96 derived from UV6139SJ cells.

FIG. 2. Effect in tumor-bearing mice of therapeutic administration ofgp96 derived from UV6139SJ cells on tumor growth measured as tumorvolume (mm³). All mice were challenged with UV6139SJ cells. The firstgroup of mice received no treatment, the second group received gp96derived form UV6139SJ cells, and the third group received gp96 derivedfrom the liver.

FIG. 3. Vaccination with cognate gp96 preparations elicits MHC classI--restricted CTLS. Mice were immunized with gp96 derived from UV6138(triangles) or UV6139SJ (rectangles) or with intact tumor cells(circles). Ten days after second immunization, spleens were removed andspleen cells were cocultered in a mixed lymphocyte tumor culture (MLTC)with irradiated tumor cells used for immunization or gp96 preparation.MLTCs were tested for cytotoxicity in a chromium release assay. Opensymbols refer to the cytotoxicity in presence of anti-MHC class Ispecific antibody K44. (A) In vitro cytotoxicity of non-immunized mice(triangle) or mice immunized with 20 microgram of gp96 derived fromUV6138 (circle) or UV6139SJ (rectangle) against targets as indicated.(B) In vitro cytotoxicity of non-immunized mice (triangle) or miceimmunized twice with 10⁷ irradiated UV6138 cells (circles) or UV6139SJcells (rectangles) against targets as indicated.

FIG. 4. Vaccination with cognate gp96 preparations elicitsradiation-resistant T cell response. Mice were immunized with gp96derived from UV6138 (triangles) or UV6139SJ (rectangles) and MLTCs setup as described in legend to FIG. 2, except that mice had beenirradiated at 400 rad twelve days after the last vaccination and MLTCswere set up three days after irradiation. Open symbols refer to thecytotoxicity in presence of anti-MHC class I specific antibody K44.

FIGS. 5A-B. FIG. 5A: ATP-bound and ATP eluted hsp70 preparation wasfound not to be associated with peptides. FIG. 5B: ADP-bound and ADPeluted hsp70 preparation was found to be associated with peptides.

5. DETAILED DESCRIPTION OP THE INVENTION

Methods and compositions for the prevention and treatment of primary andmetastatic neoplastic diseases and infectious diseases and for elicitingan immune response in a human individual, are described. The inventionis based, in part, on a newly discovered dosage regimen foradministration of compositions comprising complexes of hspsnoncovalently bound to antigenic molecules.

"Antigenic molecule" as used herein refers to the peptides with whichthe hsps are endogenously associated in vivo (e.g., in infected cells orprecancerous or cancerous tissue) as well as exogenousantigens/immunogens (i.e., with which the hsps are not complexed invivo) or antigenic/immunogenic fragments and derivatives thereof.

The methods of the invention comprise methods of eliciting an immuneresponse in an individual in whom the treatment or prevention ofinfectious diseases or cancer is desired by administering a compositioncomprising an effective amount of a complex, in which the complexconsists essentially of a hsp noncovalently bound to an antigenicmolecule. In a preferred embodiment, the complex is autologous to theindividual; that is, the complex is isolated from either from theinfected cells or the cancer cells for precancerous cells of theindividual himself (e.g., preferably prepared from infected tissues ortumor biopsies of the patient). Alternatively, the complex is producedin vitro (e.g., wherein a complex with an exogenous antigenic moleculeis desired). Alternatively, the hsp and/or the antigenic molecule can beisolated from the individual or from others or by recombinant productionmethods using a cloned hsp originally derived from the individual orfrom others. Exogenous antigens and fragments and derivatives (bothpeptide and non-peptide) thereof for use in complexing with hsps, can beselected from among those known in the art, as well as those readilyidentified by standard immunoassays know in the art by the ability tobind antibody or MHC molecules (antigenicity) or generate immuneresponse (immunogenicity). Complexes of hsps and antigenic molecules canbe isolated from cancer or precancerous tissue of a patient, or from acancer cell line, or can be produced in vitro (as is necessary in theembodiment in which an exogenous antigen is used as the antigenicmolecule).

The invention also provides a method for measuring tumor rejection invivo in an individual, preferably a human comprising measuring thegeneration by the individual of MHC Class I-restricted CD8⁺ cytotoxic Tlymphocytes specific to the tumor.

The hsps of the present invention that can be used include but are notlimited to, hsp70, hsp90, gp96 alone or in combination. Preferably, thehsps are human hsps.

Heat shock proteins, which are also referred to interchangeably hereinas stress proteins, useful in the practice of the instant invention canbe selected from among any cellular protein that satisfies any one ofthe following criteria. It is a protein whose intracellularconcentration increases when a cell is exposed to a stressful stimuli,it is capable of binding other proteins or peptides, it is capable ofreleasing the bound proteins or peptides in the presence of adenosinetriphosphate (ATP) or low pH, or it is a protein showing at least 35%homology with any cellular protein having any of the above properties.

The first stress proteins to be identified were the heat shock proteins(hsps). As their name implies, hsps are synthesized by a cell inresponse to heat shock. To date, three major families of hsp have beenidentified based on molecular weight. The families have been calledhsp60, hsp70 and hsp90 where the numbers reflect the approximatemolecular weight of the stress proteins in kilodaltons. Many members ofthese families were found subsequently to be induced in response toother stressful stimuli including, but not limited to, nutrientdeprivation, metabolic disruption, oxygen radicals, and infection withintracellular pathogens. (See Welch, May 1993, Scientific American56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science260:1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist,et al., 1988, Annu. Rev. Genetics 22:631-677), the disclosures of whichare incorporated herein by reference. It is contemplated thathsps/stress proteins belonging to all of these three families can beused in the practice of the instant invention.

The major hsps can accumulate to very high levels in stressed cells, butthey occur at low to moderate levels in cells that have not beenstressed. For example, the highly inducible mammalian hsp70 is hardlydetectable at normal temperatures but becomes one of the most activelysynthesized proteins in the cell upon heat shock (Welch, et al., 1985,J. Cell. Biol. 101:1198-1211). In contrast, hsp90 and hsp60 proteins areabundant at normal temperatures in most, but not all, mammalian cellsand are further induced by heat (Lai, et al., 1984, Mol. Cell. Biol.4:2802-10; van Bergen en Henegouwen, et al., 1987, Genes Dev. 1:525-31).

Heat shock proteins are among the most highly conserved proteins inexistence. For example, DnaK, the hsp70 from E. coli has about 50% aminoacid sequence identity with hsp70 proteins from excoriates (Bardwell, etal., 1984, Proc. Natl. Acad. Sci. 81:848-852). The hsp60 and hsp90families also show similarly high levels of intra families conservation(Hickey, et al., 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol.Cell. Biol. 9:2279-2283). In addition, it has been discovered that thehsp60, hsp70 and hsp90 families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels are notaltered by stress. Therefore it is contemplated that the definition ofstress protein, as used herein, embraces other proteins, muteins,analogs, and variants thereof having at least 35% to 55%, preferably 55%to 75%, and most preferably 75% to 85% amino acid identity with membersof the three families whose expression levels in a cell are enhanced inresponse to a stressful stimulus. The purification of stress proteinsbelonging to these three families is described below.

The immunogenic hsp-peptide complexes of the invention may include anycomplex containing an hsp and a peptide that is capable of inducing animmune response in a mammal. The peptides are preferably non covalentlyassociated with the hsp. Preferred complexes may include, but are notlimited to, hsp60-peptide, hsp70-peptide and hsp90-peptide complexes.For example, an hsp called gp96 which is present in the endoplasmicreticulum of eukaryotic cells and is related to the cytoplasmic hsp90'scan be used to generate an effective vaccine containing a gp96-peptidecomplex.

Although the hsps can be allogeneic to the patient, in a preferredembodiment, the hsps are autologous to (derived from) the patient towhom they are administered. The hsps and/or antigenic molecules can bepurified from natural sources, chemically synthesized, or recombinantlyproduced. The invention provides methods for determining doses for humancancer immunotherapy by evaluating the optimal dose of hsp noncovalentlybound to peptide complexes in experimental tumor models andextrapolating the data. Specifically, a scaling factor not exceeding afifty fold increase over the effective dose estimated in animals, isused as the optimal prescription method for cancer immunotherapy orvaccination in human subjects.

The invention provides combinations of compositions which enhance theimmunocompetence of the host individual and elicit specific immunityagainst infectious agents or specific immunity against preneoplastic andneoplastic cells. The therapeutic regimens and pharmaceuticalcompositions of the invention are described below. These compositionshave the capacity to prevent the onset and progression of infectiousdiseases and prevent the development of tumor cells and to inhibit thegrowth and progression of tumor cells indicating that such compositionscan induce specific immunity in infectious diseases and cancerimmunotherapy.

Hsps appear to induce an inflammatory reaction at the tumor site andultimately cause a regression of the tumor burden in the cancer patientstreated. Cancers which can be treated with complexes of hspsnoncovalently bound to antigenic molecules include, but are not limitedto, human sarcomas and carcinomas.

Accordingly, the invention provides methods of preventing and treatingcancer in an individual comprising administering a composition whichstimulates the immunocompetence of the host individual and elicitsspecific immunity against the preneoplastic and/or neoplastic cells. Asused herein, "preneoplastic" cell refers to a cell which is intransition from a normal to a neoplastic form; and morphologicalevidence, increasingly supported by molecular biologic studies,indicates that preneoplasia progresses through multiple steps.Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia,or most particularly, dysplasia (for review of such abnormal growthconditions (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B.Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form ofcontrolled cell proliferation involving an increase in cell number in atissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder. Although preneoplastic lesions may progress to neoplasia, theymay also remain stable for long periods and may even regress,particularly if the inciting agent is removed or if the lesion succumbsto an immunological attack by its host.

The therapeutic regimens and pharmaceutical compositions of theinvention may be used with additional immune response enhancers orbiological response modifiers including, but not limited to, thecytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF, or other cytokineaffecting immune cells. In accordance with this aspect of the invention,the complexes of the hsp and antigenic molecule are administered incombination therapy with one or more of these cytokines.

The invention further relates to administration of complexes ofhsp-antigenic molecules to individuals at enhanced risk of cancer due tofamilial history or environmental risk factors.

5.1. Dosage Regimens

It was established in experimental tumor models (Blachere et al., 1993,J. Immunotherapy 14:352-356) that the lowest dose of hsp noncovalentlybound to peptide complexes which produced tumor regression in mice wasbetween 10 and 25 microgram/mouse weighing 20-25g which is equal to 25mg/25g=1 mg/kg. Prior art methods extrapolate to human dosages based onbody weight and surface area. For example, prior art methods ofextrapolating human dosage based on body weight can be carried out asfollows: since the conversion factor for converting the mouse dosage tohuman dosage is Dose Human per kg=Dose Mouse per kg×12 (See Freireich,E. J., et al., 1966, Cancer Chemotherap. Rep. 50:219-244), the effectivedose of hsp-peptide complexes in humans weighing 70 kg should be 1mg/kg÷12×70, i.e., about 6 mg (5.8 mg).

Drug doses are also given in milligrams per square meter of body surfacearea because this method rather than body weight achieves a goodcorrelation to certain metabolic and excretionary functions (Shirkey, H.C., 1965, JAMA 193:443). Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as indicated below in Table 1 (Freireich, E.J., et al., 1966, Cancer Chemotherap. Rep. 50:219-244).

                  TABLE 1                                                         ______________________________________                                        REPRESENTATIVE SURFACE AREA TO WEIGHT                                         RATIOS (km) FOR VARIOUS SPECIES.sup.1                                                    Body weight Surface Area                                           Species    (kg)        (Sqm)     km Factor                                    ______________________________________                                        Mouse      0.02        0.0066    3.0                                          Rat        0.15        0.025     5.9                                          Monkey     3.0         0.24      12                                           Dog        8.0         0.40      20                                           Human, Child                                                                             20          0.80      25                                           Adult      60          1.6       37                                           ______________________________________                                         .sup.1 Freireich, et al., 1966, Cancer Chemotherap. Rep. 50: 219-244.    

Example: To express a mg/kg dose in any given species as the equivalentmg/sq m dose, multiply the dose by the appropriate km factor. In adulthuman, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/sq m.

In contrast to both of the above-described prior art methods ofdetermining dosage levels, the present invention provides dosages of thepurified complexes of hsps and antigenic molecules that are much smallerthan the dosages estimated by the prior art. For example, according tothe invention, an amount of hsp70- and/or gp96-antigenic moleculecomplexes is administered that is in the range of about 10 microgram toabout 600 micrograms for a human patient, the preferred human dosagebeing the same as used in a 25 g mouse, i.e., in the range of 10-100micrograms. The dosage for hsp-90 peptide complexes in a human patientprovided by the present invention is in the range of about 50 to 5,000micrograms, the preferred dosage being 100 micrograms.

The doses recited above are preferably given once weekly for a period ofabout 4-6 weeks, and the mode or site of administration is preferablyvaried with each administration. In a preferred example, subcutaneousadministrations are given, with each site of administration variedsequentially. Thus, by way of example and not limitation, the firstinjection may be given subcutaneously on the left arm, the second on theright arm, the third on the left belly, the fourth on the right belly,the fifth on the left thigh, the sixth on the right thigh, etc. The samesite may be repeated after a gap of one or more injections. Also, splitinjections may be given. Thus, for example, half the dose may be givenin one site and the other half on an other site on the same day.

Alternatively, the mode of administration is sequentially varied, e.g.,weekly injections are given in sequence subcutaneously, intramuscularly,intravenously or intraperitoneally.

After 4-6 weeks, further injections are preferably given at two-weekintervals over a period of time of one month. Later injections may begiven monthly. The pace of later injections may be modified, dependingupon the patient's clinical progress and responsiveness to theimmunotherapy.

The invention is illustrated by non-limiting examples in Sections 6 and7.

5.2. Therapeutic Compositions for Immune Responses to Cancer

The compositions comprising hsp noncovalently bound to antigenicmolecules are administered to elicit an effective specific immuneresponse to the complexed antigenic molecules (and not to the hsp).

In a preferred embodiment, non-covalent complexes of hsp70, hsp90 andgp96 with peptides are prepared and purified postoperatively from tumorcells obtained from the cancer patient.

In accordance with the methods described herein, immunogenic orantigenic peptides that are endogenously complexed to hsps or MHCantigens can be used as antigenic molecules. For example, such peptidesmay be prepared that stimulate cytotoxic T cell responses againstdifferent tumor antigens (e.g., tyrosinase, gp100, melan-A, gp75,mucins, etc.) and viral proteins including, but not limited to, proteinsof immunodeficiency virus type I (HIV-I), human immunodeficiency virustype II (HIV-II), hepatitis type A, hepatitis type B,. hepatitis type C,influenza, Varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus and polio virus. In theembodiment wherein the antigenic molecules are peptides noncovalentlycomplexed to hsps in vivo, the complexes can be isolated from cells, oralternatively, produced in vitro from purified preparations each of hspsand antigenic molecules.

In another specific embodiment, antigens of cancers (e.g., tumors) orinfectious agents (e.g., viral antigen, bacterial antigens, etc.) can beobtained by purification from natural sources, by chemical synthesis, orrecombinantly, and, through in vitro procedures such as that describedbelow, noncovalently complexed to hsps.

In an embodiment wherein the hsp-antigenic molecule complex to be usedis a complex that is produced in vivo in cells, exemplary purificationprocedures such as described in Sections 5.2.1-5.2.3 below can beemployed. Alternatively, in an embodiment wherein one wishes to useantigenic molecules by complexing to hsps in vitro, hsps can be purifiedfor such use from the endogenous hsp-peptide complexes in the presenceof ATP or low pH (or chemically synthesized or recombinantly produced).The protocols described herein may be used to isolate hsp-peptidecomplexes, or the hsps alone, from any eukaryotic cells for example,tissues, isolated cells,. or immortalized eukaryote cell lines infectedwith a preselected intracellular pathogen, tumor cells or tumor celllines.

5.2.1. Preparation and Purification of Hsp70-peptide Complexes

The purification of hsp70-peptide complexes has been describedpreviously, see, for example, Udono et al., 1993, J. Exp. Med.178:1391-1396. A procedure that may be used, presented by way of examplebut not limitation, is as follows:

Initially, tumor cells are suspended in 3 volumes of 1 X Lysis bufferconsisting of 5 mM sodium phosphate buffer, pH 7, 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 minutes and then homogenized in a douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2 X lysis buffer prior to mixing withCon A Sepharose. The supernatant is then allowed to bind to the Con ASepharose for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 minutes. Then the resulting supernatant is harvested and applied to aMono Q FPLC column equilibrated in 20 mM Tris-Acetate pH 7.5, 20 mMNaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The column is thendeveloped with a 20 mM to 500 mM NaCl gradient and then eluted fractionsfractionated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and characterized by immunoblotting using anappropriate anti-hsp70 antibody (such as from clone N27F3-4, fromStressGen).

Fractions strongly immunoreactive with the anti-hsp70 antibody arepooled and the hsp70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex® G25 column (Pharmacia). Ifnecessary the hsp70 preparation thus obtained can be repurified throughthe Mono Q FPCL Column as described above.

The hsp70-peptide complex can be purified to apparent homogeneity usingthis method. Typically 1 mg of hsp70-peptide complex can be purifiedfrom 1 g of cells/tissue.

The present invention further describes a new and rapid method forpurification of hsp70-peptide complexes. This improved method comprisescontacting cellular proteins with ADP or a nonhydrolyzable analog of ATPaffixed to a solid substrate, such that hsp70 in the lysate can bind tothe ADP or nonhydrolyzable ATP analog, and eluting the bound hsp70. Apreferred method uses column chromatography with ADP affixed to a solidsubstratum (e.g., ADP-agarose). The resulting hsp70 preparations arehigher in purity and devoid of contaminating peptides. The hsp70 yieldsare also increased significantly by about more than 10 fold.Alternatively, chromatography with nonhydrolyzable analogs of ATP,instead of ADP, can be used for purification of hsp70-peptide complexes.By way of example but not limitation, purification of hsp70-peptidecomplexes by ADP-agarose chromatography was carried out as described inExample Section 9.

5.2.2. Preparation and Purification of Hsp 90-peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

Initially, tumor cells are suspended in 3 volumes of 1 X Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 minutes and then homogenized in a douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of 2X Lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Thematerial that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 10 mM Tris-Acetate pH 7.5,0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resultingsupernatant is harvested and applied to a Mono Q FPLC columnequilibrated with lysis buffer. The proteins are then eluted with a saltgradient of 200 mM to 600 mM NaCl.

The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the hsp90-peptide complexes identified by immunoblottingusing an anti-hsp90 antibody such as 3G3 (Affinity Bioreagents).Hsp90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of hsp90-peptide complex can bepurified from 1 g of cells/tissue.

5.2.3. Preparation and Purification of gp96-peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

A pellet of tumors is resuspended in 3 volumes of buffer consisting of30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM PMSF and the cellsallowed to swell on ice 20 minutes. The cell pellet then is homogenizedin a Dounce homogenizer (the appropriate clearance of the homogenizerwill vary according to each cells type) on ice until >95% cells arelysed.

The lysate is centrifuged at 1,000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep then is recentrifuged at 100,000 g for 90 minutes. The gp96-peptidecomplex can be purified either from the 100,000 pellet or from thesupernatant.

When purified from the supernatant, the supernatant is diluted withequal volume of 2 X lysis buffer and the supernatant mixed for 2-3 hoursat 4° C. with Con a sepharose equilibrated with PBS containing 2 mM Ca²⁺and 2 mM Mg²⁺. Then, the slurry is packed into a column and washed with1 X lysis buffer until the OD₂₈₀ drops to baseline. Then, the column iswashed with 1/3 column bed volume of 10% α-methyl mannoside (α-MM)dissolved in PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the column sealedwith a piece of parafilm, and incubated at 37° C. for 15 minutes. Thenthe column is cooled to room temperature and the parafilm removed fromthe bottom of the column. Five column volumes of the α-MM buffer areapplied to the column and the eluate analyzed by SDS-PAGE. Typically theresulting material is about 60-95% pure, however this depends upon thecell type and the tissue-to-lysis buffer ratio used. Then the sample isapplied to a Mono Q FPLC column (Pharmacia) equilibrated with a buffercontaining 5 mM sodium phosphate, pH 7. The proteins then are elutedfrom the column with a 0-1 M NaCl gradient and the gp96 fraction elutesbetween 400 mM and 550 mM NaCl.

The procedure, however, may be modified by two additional steps, usedeither alone or in combination, to consistently produce apparentlyhomogeneous gp96-peptide complexes. One optional step involves anammonium sulfate precipitation prior to the Con A purification step andthe other optional step involves DEAE-Sepharose purification after theCon A purification step but before the Mono Q FPLC step.

In the first optional step, the supernatant resulting from the 100,000 gcentrifugation step is brought to a final concentration of 50% ammoniumsulfate by the addition of ammonium sulfate. The ammonium sulfate isadded slowly while gently stirring the solution in a beaker placed in atray of ice water. The solution is stirred from about 1/2 to 12 hours at4° C. and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34rotor). The supernatant resulting from this step is removed, brought to70% ammonium sulfate saturation by the addition of ammonium sulfatesolution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor). Theresulting pellet from this step is harvested and suspended in PBScontaining 70% ammonium sulfate in order to rinse the pellet. Thismixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pelletdissolved in PBS containing 2 mM Ca²⁺ and Mg²⁺. Undissolved material isremoved by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor).Then, the solution is mixed with Con A Sepharose and the procedurefollowed as before.

In the second optional step, the gp96 containing fractions eluted fromthe Con A column are pooled and the buffer exchanged for 5 mM sodiumphosphate buffer, pH 7, 300 mM NaCl by dialysis, or preferably by bufferexchange on a Sephadex G25 column. After buffer exchange, the solutionis mixed with DEAB-Sepharose previously equilibrated with 5 mM sodiumphosphate buffer, pH 7, 300 mM NaCl. The protein solution and the beadsare mixed gently for 1 hour and poured into a column. Then, the columnis washed with 5 mM sodium phosphate buffer, pH 7, 300 mM NaCl, untilthe absorbance at 280 nM drops to baseline. Then, the bound protein iseluted from the column with five volumes of 5 mM sodium phosphatebuffer, pH 7, 700 mM NaCl. Protein containing fractions are pooled anddiluted with 5 mM sodium phosphate buffer, pH 7 in order to lower thesalt concentration to 175 mM. The resulting material then is applied tothe Mono Q FPLC column (Pharmacia) equilibrated with 5 mM sodiumphosphate buffer, pH 7 and the protein that binds to the Mono Q FPLCcolumn (Pharmacia) is eluted as described before.

It is appreciated, however, that one skilled in the art may assess, byroutine experimentation, the benefit of incorporating the secondoptional step into the purification protocol. In addition, it isappreciated also that the benefit of adding each of the optional stepswill depend upon the source of the starting material.

When the gp96 fraction is isolated from the 100,000 g pellet, the pelletis suspended in 5 volumes of PBS containing either 1% sodiumdeoxycholate or 1% octyl glucopyranoside (but without the Mg²⁺ and Ca²⁺)and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against severalchanges of PBS (also without the Mg²⁺ and Ca²⁺) to remove the detergent.The dialysate is centrifuged at 100,000 g for 90 minutes, thesupernatant harvested, and calcium and magnesium are added to thesupernatant to give final concentrations of 2 mM, respectively. Then thesample is purified by either the unmodified or the modified method forisolating gp96-peptide complex from the 100,000 g supernatant, seeabove.

The gp96-peptide complexes can be purified to apparent homogeneity usingthis procedure. About 10-20 μg of gp96 can be isolated from 1 gcells/tissue.

Infectious Disease

In an alternative embodiment wherein it is desired to treat a patienthaving an infectious disease the above-described methods in Sections5.2.1-5.2.3 are used to isolate hsp-peptide complexes from cellsinfected with an infectious organism, e.g., of a cell line or from apatient. Such infectious organisms include but are not limited to,viruses, bacterial, protozoa, fungi, and parasites as described indetail in Section 5.2.4 below.

5.2.4. Isolation of Antigenic/Immunogenic Components

It has been found that antigenic peptides and/or components can beeluted from hsp-complexes either in the presence of ATP or low pH. Theseexperimental conditions may be used to isolate peptides and/or antigeniccomponents from cells which may contain potentially useful antigenicdeterminants. Once isolated, the amino acid sequence of each antigenicpeptide may be determined using conventional amino acid sequencingmethodologies. Such antigenic molecules can then be produced by chemicalsynthesis or recombinant methods, purified, and complexed to hsps invitro.

Similarly, it has been found that potentially immunogenic peptides maybe eluted from MHC-peptide complexes using techniques well know in theart (Falk, K. et al., 1990 Nature 348:248-251; Elliott, T., et al.,1990, Nature 348:195-197; Falk, K., et al., 1991, Nature 351:290-296).

Thus, potentially immunogenic or antigenic peptides may be isolated fromeither endogenous stress protein-peptide complexes or endogenousMHC-peptide complexes for use subsequently as antigenic molecules, bycomplexing in vitro to hsps. Exemplary protocols for isolating peptidesand/or antigenic components from either of the these complexes are setforth below in Sections 5.2.4.1 and 5.2.4.2.

5.2.4.1 Peptides From Stress Protein-Peptide Complexes

Two methods may be used to elute the peptide from a stressprotein-peptide complex. One approach involves incubating the stressprotein-peptide complex in the presence of ATP. The other approachinvolves incubating the complexes in a low pH buffer.

Briefly the complex of interest is centrifuged through a Centricon 10assembly (Millipore) to remove any low molecular weight material looselyassociated with the complex. The large molecular weight fraction may beremoved and analyzed by SDS-PAGE while the low molecular weight may beanalyzed by HPLC as described below. In the ATP incubation protocol, thestress protein-peptide complex in the large molecular weight fraction isincubated with 10 mM ATP for 30 minutes at room temperature. In the lowpH protocol, acetic acid or trifluoroacetic acid is added to the stressprotein-peptide complex to give a final concentration of 10% (vol/vol)and the mixture incubated at room temperature or in a boiling water bathor any temperature in between, for 10 minutes (See, Van Bleek, et al.,1990, Nature 348:213-216; and Li, et al., 1993, EMBO Journal12:3143-3151).

The resulting samples are centrifuged through an Centricon 10 assemblyas mentioned previously. The high and low molecular weight fractions arerecovered. The remaining large molecular weight stress protein-peptidecomplexes can be reincubated with ATP or low pH to remove any remainingpeptides.

The resulting lower molecular weight fractions are pooled, concentratedby evaporation and dissolved in 0.1% trifluoroacetic acid (TFA). Thedissolved material is then fractionated by reverse phase high pressureliquid chromatography (HPLC) using for example a VYDAC™ (SeparationsGroup, Inc., Hesperia, Calif.) CIB reverse phase column equilibratedwith 0.1% TFA. The bound material is then eluted at a flow rate of about0.8 ml/min by developing the column with a linear gradient of 0 to 80%acetonitrile in 0.1% TFA. The elution of the peptides can be monitoredby OD₂₁₀ and the fractions containing the peptides collected.

5.2.4.2 Peptides from MHC-peptide Complexes.

The isolation of potentially immunogenic peptides from MHC molecules iswell known in the art and so is not described in detail herein (See,Falk, et al., 1990, Nature 348:248-251; Rotzsche, at al., 1990, Nature348:252-254; Elliott, et al., 1990, Nature 348:191-197; Falk, et al.,1991, Nature 351:290-296; Demotz, et al., 1989, Nature 343:682-684;Rotzsche, et al., 1990, Science 249:283-287), the disclosures of whichare incorporated herein by reference.

Briefly, MHC-peptide complexes may be isolated by a conventionalimmunoaffinity procedure. The peptides then may be eluted from theMHC-peptide complex by incubating the complexes in the presence of about0.1% TFA in acetonitrile. The eluted peptides may be fractionated andpurified by reverse phase HPLC, as before.

The amino acid sequences of the eluted peptides may be determined eitherby manual or automated amino acid sequencing techniques well known inthe art. Once the amino acid sequence of a potentially protectivepeptide has been determined the peptide may be synthesized in anydesired amount using conventional peptide synthesis or other protocolswell known in the art.

Peptides having the same amino acid sequence as those isolated above maybe synthesized by solid-phase peptide synthesis using procedures similarto those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149.During synthesis, N-α-protected amino acids having protected side chainsare added stepwise to a growing polypeptide chain linked by itsC-terminal and to an insoluble polymeric support i.e., polystyrenebeads. The peptides are synthesized by linking an amino group of anN-α-deprotected amino acid to an α-carboxy group of an N-α-protectedamino acid that has been activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc which is acid labile and Fmocwhich is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arethen cleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein (See, Atherton, etal., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRLPress, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2ndEd., Springer-Verlag).

Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

5.2.5 Exogenous Antigenic Molecules

Antigens or antigenic portions thereof can be selected for use asantigenic molecules, for complexing to hsps, from among those known inthe art or determined by immunoassay to be able to bind to antibody orMHC molecules (antigenicity) or generate immune response(immunogenicity). To determine immunogenicity or antigenicity bydetecting binding to antibody, various immunoassays known in the art canbe used, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), "sandwich" immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, invivo immunoassays (using colloidal gold, enzyme or radioisotope labels,for example), western blots, immunoprecipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are envisioned for use. In one embodiment for detectingimmunogenicity, T cell-mediated responses can be assayed by standardmethods, e.g., in vitro cytoxicity assays or in vivo delayed-typehypersensitivity assays.

Potentially useful antigens or derivatives thereof for use as antigenicmolecules can also be identified by various criteria, such as theantigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby, 1985, Summary, in Vaccines 85, Lerner, et al. (eds.), ColdSpring Harbor Laboratory, Cold Spring Harbor, New York, pp. 388-389),type or group specificity, recognition by patients' antisera or immunecells, and/or the demonstration of protective effects of antisera orimmune cells specific for the antigen. In addition, where it is desiredto treat or prevent a disease caused by pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.

Preferably, where it is desired to treat or prevent cancer, knowntumor-specific antigens or fragments or derivatives thereof are used.For example, such tumor specific or tumor-associated antigens includebut are not is limited to KS 1/4 pan-carcinoma antigen (Perez andWalker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma7(4):407-415); ovarian carcinoma antigen (CA125) (Yu, et al., 1991,Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailer, et al.,1990, Nucl. Acids Res. 18(16):4928); prostate specific antigen (Henttuand Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli,et al., 1993, Cancer Res. 53:227-230); melanoma-associated antigen p97(Estin, et al., 1989, J. Natl. Cancer Inst. 81(6):445-446); melanomaantigen gp75 (Vijayasardahl, et al., 1990, J. Exp. Med.171(4):1375-1380); high molecular weight melanoma antigen (Natali, etal., 1987, Cancer 59:55-63) and prostate specific membrane antigen.

In a specific embodiment, an antigen or fragment or derivaltive thereofspecific to a certain tumor is selected for complexing to hsp andsubsequent administration to a patient having that tumor.

Preferably, where it is desired to treat or prevent viral diseases,molecules comprising epitopes of known viruses are used. For example,such antigenic epitopes may be prepared from viruses including, but notlimited to, hepatitis type A hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Preferably, where it is desired to treat or prevent bacterialinfections, molecules comprising epitopes of known bacteria are used.For example, such antigenic epitopes may be prepared from bacteriaincluding, but not limited to, mycobacteria rickettsia, mycoplasma,neisseria and legionella.

Preferably, where it is desired to treat or prevent protozoalinfectious, molecules comprising epitopes of known protozoa are used.For example, such antigenic epitopes may be prepared from protozoaincluding, but not limited to, leishmania, kokzidioa, and trypanosoma.

Preferably, where it is desired to treat or prevent parasiticinfectious, molecules comprising epitopes of known parasites are used.For example, such antigenic epitopes may be from parasites including,but not limited to, chlamydia and rickettsia.

5.2.6 In Vitro Production of Stress Protein-Antigenic Molecule Complexes

In an embodiment in which complexes of hsps and the peptides with whichthey are endogenously associated in vivo are not employed, complexes ofhsps to antigenic molecules are produced in vitro. As will beappreciated by those skilled in the art, the peptides either isolated bythe aforementioned procedures or chemically synthesized or recombinantlyproduced may be reconstituted with a variety of naturally purified orrecombinant stress proteins in vitro to generate immunogenicnon-covalent stress protein-antigenic molecule complexes. Alternatively,exogenous antigens or antigenic/immunogenic fragments or derivativesthereof can be noncovalently complexed to stress proteins for use in theimmunotherapeutic or prophylactic vaccines of the invention. Apreferred, exemplary protocol for noncovalently complexing a stressprotein and an antigenic molecule in vitro is discussed below.

Prior to complexing, the hsps are pretreated with ATP or low pH toremove any peptides that may be associated with the hsp of interest.When the ATP procedure is used, excess ATP is removed from thepreparation by the addition of apyranase as described by Levy, et al.,1991, Cell 67:265-274. When the low pH procedure is used, the buffer isreadjusted to neutral pH by the addition of pH modifying reagents.

The antigenic molecules (1 μg) and the pretreated hsp (9 μg) are admixedto give an approximately 5 antigenic molecule: 1 stress protein molarratio. Then, the mixture is incubated for 15 minutes to 3 hours at 4° to45° C. in a suitable binding buffer such as one containing 20 mM sodiumphosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl₂ and 1 mM phenyl methylsulfonyl fluoride (PMSF). The preparations are centrifuged throughCentricon 10 assembly (Millipore) to remove any unbound peptide. Theassociation of the peptides with the stress proteins can be assayed bySDS-PAGE. This is the preferred method for in vitro complexing ofpeptides isolated from MHC-peptide complexes of peptides disassociatedfrom endogenous hsp-peptide complexes.

In an alternative embodiment of the invention, preferred for producingcomplexes of hsp70 to exogenous antigenic molecules such as proteins,5-10 micrograms of purified hsp is incubated with equimolar quantitiesof the antigenic molecule in 20 mM sodium phosphate buffer pH 7.5, 0.5 MNaCl, 3 mM MgCl₂ and 1 mM ADP in a volume of 100 microliter at 37° C.for 1 hr. This incubation mixture is further diluted to 1 ml inphosphate-buffered saline.

In an alternative embodiment of the invention, preferred for producingcomplexes of gp96 or hsp90 to peptides, 5-10 micrograms of purified gp96or hsp90 is incubated with equimolar or excess quantities of theantigenic peptide in a suitable buffer such as one containing 20 mMsodium phosphate buffer pH 7.5, 0.5 M NaCl, 3nM MgC12 at 60-65° C. for5-20 min. This incubation mixture is allowed to cool to room temperatureand centrifuged one or more times if necessary, through Centricon 10assembly (Millipore) to remove any unbound peptide.

Following complexing, the immunogenic stress protein-antigenic moleculecomplexes can optionally be assayed in vitro using for example the mixedlymphocyte tumor cell assay (MLTC) described below. Once immunogeniccomplexes have been isolated they can be optionally characterizedfurther in animal models using the preferred administration protocolsand excipients discussed below.

5.2.7 Determination of Immunogenicity of Stress Protein-PeptideComplexes

The purified stress protein-antigenic molecule complexes can be assayedfor immunogenicity using the mixed lymphocyte tumor culture assay (MLTC)well known in the art.

By way of example but not limitation, the following procedure can beused. Briefly, mice are injected subcutaneously with the candidatestress protein-antigenic molecule complexes. Other mice are injectedwith either other stress protein peptide complexes or whole infectedcells which act as positive controls for the assay. The mice areinjected twice, 7-10 days apart. Ten days after the last immunization,the spleens are removed and the lymphocytes released. The releasedlymphocytes may be restimulated subsequently in vitro by the addition ofdead cells that expressed the complex of interest.

For example, 8×10⁶ immune spleen cells may be stimulated with 4×10⁴mitomycin C treated or γ-irradiated (5-10,000 rads) infected cells (orcells transfected with an appropriate gene, as the case may be) in 3 mlRPMI medium containing 10% fetal calf serum. In certain cases 33%secondary mixed lymphocyte culture supernatant may be included in theculture medium as a source of T cell growth factors (See, Glasebrook, etal., 1980, J. Exp. Med. 151:876). To test the primary cytotoxic T cellresponse after immunization, spleen cells may be cultured withoutstimulation. In some experiments spleen cells of the immunized mice mayalso be restimulated with antigenically distinct cells, to determine thespecificity of the cytotoxic T cell response.

Six days later the cultures are tested for cytotoxicity in a 4 hour ⁵¹Cr-release assay (See, Palladino, et al., 1987, Cancer Res. 47:5074-5079and Blachere, at al., 1993, J. Immunotherapy 14:352-356). In this assay,the mixed lymphocyte culture is added to a target cell suspension togive different effector:target (E:T) ratios (usually 1:1 to 40:1). Thetarget cells are prelabelled by incubating 1×10⁶ target cells in culturemedium containing 200 mCi ⁵¹ Cr/ml for one hour at 37° C. The cells arewashed three times following labeling. Each assay point (E:T ratio) isperformed in triplicate and the appropriate controls incorporated tomeasure spontaneous ⁵¹ Cr release (no lymphocytes added to assay) and100% release (cells lysed with detergent). After incubating the cellmixtures for 4 hours, the cells are pelleted by centrifugation at 200 gfor 5 minutes. The amount of ⁵¹ Cr released into the supernatant ismeasured by a gamma counter. The percent cytotoxicity is measured as cpmin the test sample minus spontaneously released cpm divided by the totaldetergent released cpm minus spontaneously released cpm.

In order to block the MHC class I cascade a concentrated hybridomasupernatant derived from K-44 hybridoma cells (an anti-MHC class Ihybridoma) is added to the test samples to a final concentration of12.5%.

5.3. Formulation

Hsp-antigenic molecule complexes of the invention may be formulated intopharmaceutical preparations for administration to mammals for treatmentor prevention of cancer or infectious diseases. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may be prepared, packaged, and labelled for treatment of theindicated tumor, such as human sarcomas and carcinomas, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease. Alternatively, it can be labeled for treatment of theappropriate infectious disease. Alternatively, pharmaceuticalcompositions may be formulated for treatment of appropriate infectiousdiseases.

If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions. Alternatively, if the resultingcomplex has poor solubility in aqueous solvents, then it may beformulated with a non-ionic surfactant such as Tween, or polyethyleneglycol. Thus, the compounds and their physiologically acceptablesolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral, rectal administration or, in the case of tumors, directlyinjected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of thehsp-antigenic molecule complexes in pharmaceutically acceptable form.The hsp-antigenic molecule. complex in a vial of a kit of the inventionmay be in the form of a pharmaceutically acceptable solution, e.g., incombination with sterile saline, dextrose solution, or bufferedsolution, or other pharmaceutically acceptable sterile fluid.Alternatively, the complex may be lyophilized or desiccated; in thisinstance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of hsp-antigenic molecule complexes by aclinician or by the patient.

5.4 Target Infectious Diseases

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents including, but notlimited to, viruses, bacteria, fungi, protozoa and parasites.

Viral diseases that can be treated or prevented by the methods of thepresent invention include, but are not limited to, those caused byhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus; rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Bacterial diseases that can be treated or prevented by the methods ofthe present invention are caused by bacteria including, but not limitedto, mycobacteria rickettsia, mycoplasma, neisseria and legionella.

Protozoal diseases that can be treated or prevented by the methods ofthe present invention are caused by protozoa including, but not limitedto, leishmania, kokzidioa, and trypanosoma.

Parasitic diseases that can be treated or prevented by the methods ofthe present invention are caused by parasites including, but not limitedto, chlamydia and rickettsia.

5.5. Target Cancers

Cancers that can be treated or prevented by the methods of the presentinvention include, but not limited to human sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphorytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease. Specific examples of such cancers are described in thesections below.

In a specific embodiment the cancer is metastatic. In another specificembodiment, the patient having a cancer is immunosuppressed by reason ofhaving undergone anti-cancer therapy (e.g., chemotherapy radiation)prior to administration of the hsp-antigenic molecule complexes of theinvention.

5.5.1. Colorectal Cancer Metastatic to the Liver

In 1992, approximately 150,000 Americans were diagnosed with colorectalcancer and more than 60,000 died as a result of colorectal metastases.At the time of their deaths, 80 percent of patients with colorectalcancer have metastatic disease involving the liver, and one-half ofthese patients have no evidence of other (extrahepatic) metastases. Mostmetastatic tumors of the liver are from gastrointestinal primaries.Unfortunately, the natural history of metastatic liver lesions carries agrave prognosis and systemic chemotherapy regimens have been unable toinduce significant response rates or alter length of survival (Drebin,J. A., et al., in Current Therapy In Oncology, ed. J. E. Niederhuber, B.C. Decker, Mosby, 1993, p.426).

Colorectal cancer initially spreads to regional lymph nodes and thenthrough the portal venous circulation to the liver, which represents themost common visceral site of metastasis. The symptoms that lead patientswith colorectal cancer to seek medical care vary with the anatomicallocation of the lesion. For example, lesions in the ascending colonfrequency ulcerate, which leads to chronic blood loss in the stool.

Radical resection offers the greatest potential for cure in patientswith invasive colorectal cancer. Before surgery, the CEA titer isdetermined. Radiation therapy and chemotherapy are used in patients withadvanced colorectal cancer. Results with chemotherapeutic agents (e.g.,5-fluorouracil) are mixed and fewer than 25 percent of patientsexperience a greater than 50 percent reduction in tumor mass (Richards,2d., F., et al., 1986, J. Clin. Oncol. 4:565).

Patients with widespread metastases have limited survival and systemicchemotherapy has little impact in this group of patients. In addition,systemically administered chemotherapy is often limited by the severityof toxicities associated with the various agents, such as severediarrhea, mucositis and/or myelosuppression. Other techniques, includinghepatic radiation, systemic chemotherapy, hepatic arterial ligation,tumor embolization and immunotherapy have all been explored, but, forthe most part, have proven ineffectual in prolonging patient survival.

In a specific embodiment, the present invention provides compositionsand methods for enhancing tumor specific immunity in individualssuffering from colorectal cancer metastasized to the liver, in order toinhibit the progression of the neoplastic disease. Preferred methods oftreating these neoplastic diseases comprise administering a compositionof autologous hsp noncovalently bound to peptide complexes, whichelicits tumor-specific immunity against the tumor cells. Mostspecifically, the use of a composition of the invention, comprisinggp96, can result in nearly complete inhibition of liver cancer growth incancer patients, without inducing toxicity and thus providing a dramatictherapeutic effect.

Accordingly, as an example of the method of the invention, gp96 isadministered to a patient diagnosed with colorectal cancer, with orwithout liver metastasis, via one of many different routes ofadministration, the preferred routes being subcutaneous at differentanatomical sites, e.g., left arm, right arm, left belly, right belly,left thigh, right thigh, etc. These routes of administration are used insequence and the site of injection is varied for each weekly injectionas described in Section 7. The preparations and use of therapeuticallyeffective compositions for the prevention and treatment of primary andmetastatic cancers are described in detail in the sections which followand by way of example, infra.

5.5.2. Hepatocellular Carcinoma

Hepatocellular carcinoma is generally a disease of the elderly in theUnited States. Although many factors may lead to hepatocellularcarcinoma, the disease is usually limited to those persons withpreexisting liver disease. Approximately 60 to 80 percent of patients inthe United States with hepatocellular carcinoma have a cirrhotic liverand about four percent of individuals with a cirrhotic liver eventuallydevelop hepatocellular carcinoma (Niederhuber, J. E., (ed.), 1993,Current Therapy in Oncology, B. C. Decker, Mosby). The risk is highestin patients whose liver disease is caused by inherited hemochromatosisor hepatic B viral infection (Bradbear, R. A., et al., 1985, J. Natl.Cancer Inst. 75:81; Beasley, R. P., et al., 1981, Lancet 2:1129). Othercauses of cirrhosis that can lead to hepatocellular carcinoma includealcohol abuse and hepatic fibrosis caused by chronic administration ofmethotrexate. The most frequent symptoms of hepatocellular carcinoma arethe development of a painful mass in the right upper quadrant orepigastrium, accompanied by weight loss. In patients with cirrhosis, thedevelopment of hepatocellular carcinoma is preceded by ascites, portalhypertension and relatively abrupt clinical deterioration. In mostcases, abnormal values in standard liver function tests such as serumaminotransferase and alkaline phosphatase are observed.

CT scans of the liver are used to determine the anatomic distribution ofhepatocellular carcinoma and also provide orientation for percutaneousneedle biopsy. Approximately 70 percent of patients with hepatocellularcarcinoma have an elevated serum alpha-fetoprotein concentration(McIntire, K. R., et al., 1975, Cancer Res. 35:991) and itsconcentration correlates with the extent of the disease.

Radical resection offers the only hope for cure in patients withhepatocellular carcinoma. Such operative procedures are associated withfive-year survival rates of 12 to 30 percent. Liver transplantation mayimprove survival of some younger individuals. However, most patients arenot surgical candidates because of extensive cirrhosis multifocal tumorpattern or scarcity of compatible donor organs. Chemotherapeutic agentshave been administered either by intravenous route or through anintrahepatic arterial catheter. Such therapy has sometimes been combinedwith irradiation to the liver. Reductions in the size of measurabletumors of 50% or more have been reported in some patients treated witheither systemic doxorubicin or 5-fluorouracil. However, chemotherapyoften induces immunosuppression and rarely causes the tumor to disappearcompletely and the duration of response is short. The prognosis forpatients with hepatocellular carcinoma is negatively correlated withcirrhosis and metastases to the lungs or bone. Median survival forpatients is only four to six months. In another specific embodiment, thepresent invention provides compositions and methods for enhancingspecific immunity in individuals suffering from hepatocellular carcinomain order to inhibit the progression of the neoplastic disease andultimately eradicate all preneoplastic and neoplastic cells.

5.5.3. Breast Cancer

Another specific aspect of the invention relates to the treatment ofbreast cancer. The American Cancer Society estimated that in 1992180,000 American women were diagnosed with breast cancer and 46,000succumbed to the disease (Niederhuber, J. E. ed. Current Therapy inOncology B. C. Decker, Mosby, 1993). This makes breast cancer the secondmajor cause of cancer death in women, ranking just behind lung cancer. Adisturbing fact is the observation that breast cancer has beenincreasing at a rate of 3 percent per year since 1980 (Niederhuber, J.E., ed. Current Therapy in Oncology, B. C. Decker, Mosby, (1993)). Thetreatment of breast cancer presently involves surgery, radiation,hormonal therapy and/or chemotherapy. Consideration of two breast cancercharacteristics, hormone receptors and disease extent, has governed howhormonal therapies and standard-dose chemotherapy are sequenced toimprove survival and maintain or improve quality of life. A wide rangeof multidrug regimens have been used as adjuvant therapy in breastcancer patients, including, but not limited to combinations of 2cyclophosphamide, doxorubicin, vincristine methotrexate, 5-fluorouraciland/or leucovorin. In a specific embodiment, the present inventionprovides hsp compositions and methods for enhancing specific immunity topreneoplastic and neoplastic mammary cells in women. The presentinvention also provides compositions and methods for preventing thedevelopment of neoplastic cells in women at enhanced risk for breastcancer, and for inhibiting cancer cell proliferation and metastasis.These compositions can be applied alone or in combination with eachother or with biological response modifiers.

5.6. Autologous Embodiment

The specific immunogenicity of hsps derives not from hsps per se, butfrom the peptides bound to them. In a preferred embodiment of theinvention directed to the use of autologous complexes of hsp-peptides ascancer vaccines, two of the most intractable hurdles to cancerimmunotherapy are circumvented. First is the possibility that humancancers, like cancers of experimental animals, are antigenicallydistinct. In an embodiment of the present invention, hsps chaperoneantigenic peptides of the cancer cells from which they are derived andcircumvent this hurdle. Second, most current approaches to cancerimmunotherapy focus on determining the CTL-recognized epitopes of cancercell lines. This approach requires the availability of cell lines andCTLs against cancers. These reagents are unavailable for an overwhelmingproportion of human cancers. In an embodiment of the present inventiondirected to autologous complexes of hsps and peptides, cancerimmunotherapy does not depend on the availability of cell lines or CTLsnor does it require definition of the antigenic epitopes of cancercells. These advantages make autologous hsps noncovalently bound topeptide complexes attractive and novel immunogens against cancer.

5.7. Prevention and Treatment of Primary and Metastatic NeoplasticDiseases

There are many reasons why immunotherapy as provided by the presentinvention is desired for use in cancer patients. First, if cancerpatients are immunosuppressed and surgery, with anesthesia, andsubsequent chemotherapy, may worsen the immunosuppression, then withappropriate immunotherapy in the preoperative period, thisimmunosuppression may be prevented or reversed. This could lead to fewerinfectious complications and to accelerated wound healing. Second, tumorbulk is minimal following surgery and immunotherapy is most likely to beeffective in this situation. A third reason is the possibility thattumor cells are shed into the circulation at surgery and effectiveimmunotherapy applied at this time can eliminate these cells.

The preventive and therapeutic methods of the invention are directed atenhancing the immunocompetence of the cancer patient either beforesurgery, at or after surgery, and to induce tumor-specific immunity tocancer cells, with the objective being inhibition of cancer, and withthe ultimate clinical objective being total cancer regression anderadication.

5.8. Monitoring of Effects During Cancer Prevention and Immunotherapywith Hsp-peptide Complexes

The effect of immunotherapy with hsp-antigenic molecule complexes ondevelopment and progression of neoplastic diseases can be monitored byany methods known to one skilled in the art, including but not limitedto measuring: a) delayed hypersensitivity as an assessment of cellularimmunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels oftumor specific antigens, e.g., carcinoembryonic (CEA) antigens; d)changes in the morphology of tumors using techniques such as a computedtomographic (CT) scan; and e) changes in levels of putative biomarkersof risk for a particular cancer in individuals at high risk, and f)changes in the morphology of tumors using a sonogram.

5.8.1. Delayed Hypersensitivity Skin Test

Delayed hypersensitivity skin tests are of great value in the overallimmunocompetence and cellular immunity to an antigen. Inability to reactto a battery of common skin antigens is termed anergy (Sato, T., et al,1995, Clin. Immunol. Pathol. 74:.35-43).

Proper technique of skin testing requires that the antigens be storedsterile at 4° C., protected from light and reconstituted shortly beforeuse. A 25- or 27-gauge needle ensures intradermal, rather thansubcutaneous, administration of antigen. Twenty-four and 48 hours afterintradermal administration of the antigen, the largest dimensions ofboth erythema and induration are measured with a ruler. Hypoactivity toany given antigen or group of antigens is confirmed by testing withhigher concentrations of antigen or, in ambiguous circumstances, by arepeat test with an intermediate concentration.

5.8.2. Activity of Cytolytic T-lymptocytes In Vitro

8×10⁶ Peripheral blood derived T lymphocytes isolated by theFicoll-Hypaque centrifugation gradient technique, are restimulated with4×10⁴ mitomycin C treated tumor cells in 3 ml RPMI medium containing 10%fetal calf serum. In some experiments, 33% secondary mixed lymphocyteculture supernatant or IL-2, is included in the culture medium as asource of T cell growth factors.

In order to measure the primary response of cytolytic T-lymphocytesafter immunization, T cells are cultured without the stimulator tumorcells. In other experiments, T cells are restimulated with antigenicallydistinct cells. After six days, the cultures are tested for cytotoxityin a 4 hour ⁵¹ Cr-release assay. The spontaneous ⁵¹ Cr-release of thetargets should reach a level less than 20%. For the anti-MHC class Iblocking activity, a tenfold concentrated supernatant of W6/32 hybridomais added to the test at a final concentration of 12.5% (Heike M., etal., J. Immunotherapy 15:165-174).

5.8.3. Levels of Tumor Specific Antigens

Although it may not be possible to detect unique tumor antigens on alltumors, many tumors display antigens that distinguish them from normalcells. The monoclonal antibody reagents have permitted the isolation andbiochemical characterization of the antigens and have been invaluablediagnostically for distinction of transformed from nontransformed cellsand for definition of the cell lineage of transformed cells. Thebest-characterized human tumor-associated antigens are the oncofetalantigens. These antigens are expressed during embryogenesis, but areabsent or very difficult to detect in normal adult tissue. The prototypeantigen is carcinoembryonic antigen (CEA), a glycoprotein found on fetalgut an human colon cancer cells, but not on normal adult colon cells.Since CEA is shed from colon carcinoma cells and found in the serum, itwas originally thought that the presence of this antigen in the serumcould be used to screen patients for colon cancer. However, patientswith other tumors, such as pancreatic and breast cancer, also haveelevated serum levels of CEA. Therefore, monitoring the fall and rise ofCEA levels in cancer patients undergoing therapy has proven useful forpredicting tumor progression and responses to treatment.

Several other oncofetal antigens have been useful for diagnosing andmonitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulinnormally secreted by fetal liver and yolk sac cells, is found in theserum of patients with liver and germinal cell tumors and can be used asa matter of disease status.

5.8.4. Computed Tomographic (CT) Scan

CT remains the choice of techniques for the accurate staging of cancers.CT has proved more sensitive and specific than any other imagingtechniques for the detection of metastases.

5.8.5. Measurement of Putative Biomarkers

The levels of a putative biomarker for risk of a specific cancer aremeasured to monitor the effect of hsp noncovalently bound to peptidecomplexes. For example, in individuals at enhanced risk for prostatecancer, serum prostate-specific antigen (PSA) is measured by theprocedure described by Brawer, M. K., et. al., 1992, J. Urol.147:841-845, and Catalona, W. J., et al., 1993, JAMA 270:948-958; or inindividuals at risk for colorectal cancer CEA is measured as describedabove in Section 4.5.3; and in individuals at enhanced risk for breastcancer, 16-α-hydroxylation of estradiol is measured by the proceduredescribed by Schneider, J. et al., 1982, Proc. Natl. Acad. Sci. ISA79:3047-3051. The references cited above are incorporated by referenceherein in their entirety.

5.8.6. Sonogram

A Sonogram remains an alternative choice of technique for the accuratestaging of cancers.

6. EXAMPLE ADMINISTRATION OF HSP-PEPTIDE COMPLEXES IN TWO UV-INDUCEDCARCINOMA MODELS IN MICE

a) Tumor models:

Two UV-induced carcinomas were studied in the C3H/HeN mice (Ward, etal., 1989, J. Exp. Med. 170:217): (i) the highly immunogenic UV6138carcinoma, and (ii) the less immunogenic UV6139ST carcinoma.

b) Gp96 preparations were prepared from the UV6138 and UV6139SJcarcinomas by the procedures described above in Section 5.2.3. The gp96preparations were administered without adjuvants.

6.1 Prevention Modality

(a) Materials and Method:

The ability of gp96 preparations to prevent development of UV-inducedcarcinoma was tested. A total of six groups of female C₃ H/HeN mice(obtained from the National Cancer Institute, Frederick, Md.), weighingapproximately 25 g each, were used. Two groups of mice were given twiceat a ten day interval, either (i) phosphate buffer saline (PBS), (ii) 25microgram/mouse of gp96 derived from UV6138 carcinomas, or (iii) 25microgram/mouse of gp96 derived from UV3169SJ carcinoma.

In each set, mice were challenged with 10⁷ cells from either the UV6138carcinoma or the UV6139SJ carcinoma 15 days after the second injectionwith PBS or gp96. Tumors were measured at 2 day intervals. Since theUV6138 tumor is a regressor tumor, mice were irradiated at 400 rad 10days after the second injection with PBS or gp96 in order to permitgrowth of the tumor. The UV6139SJ challenged mice were not irradiated.

b) Results

Administration of gp96 isolated from the UV6138 carcinoma rendered themice immune to the UV6138 challenge but not the UV6139SJ challenge (FIG.1). Conversely, administration of gp96 isolated from the UV6139SJconferred resistance to the UV6139SJ cells but not to the UV6138 cells.The resistance rendered by the gp96 derived from the UV6138 against theUV6138 cells was much greater (6 out of 7 mice) than the resistancerendered by the gp96 derived from the UV6139SJ against the UV6139SJcells (2 out of 4 mice) (FIG. 1). These results indicate thatadministration of gp96 preparations derived from the two UV-inducedcarcinomas immunized syngeneic mice from the respective cancer cell typeand that the resistance rendered was greater and more uniform againstthe more immunogenic carcinoma cells.

6.2 Treatment Modality

a) Materials and Methods

The ability of gp96 preparations to mediate therapy of pre-existingcancers was tested. Three groups of mice were injected intradermallywith 10⁷ cells of the UV6139SJ carcinoma. The mice were kept underobservation until the tumors became visible and palpable at day 4.Thereafter, the mice in the first group received no treatment, eachmouse in the second group received every other day for a total of 5injections of 6 micrograms each of gp96 derived from the UV6139SJcarcinoma cells, and each mouse in the third group received in a similarmanner a total of 5 injections of gp96 derived from the normal liver.

b) Results

Tumor growth monitored as diameter width, was significantly retarded inmice treated with tumor-derived gp96 but not in mice treated with theliver-derived gp96 or in the untreated mice (FIG. 2). These resultsindicated a therapeutic effect of gp96-complexes in the UV6139SJcarcinoma model. All mice eventually succumbed to tumor growth. Ascrutiny of the kinetics of tumor growth in treated and controlled miceshows that administration of tumor-derived gp96 had an immediateinhibitory effect on tumor growth and that the effect appears to havediminished after treatment with gp96 was terminated.

6.3 Measuring Generation of MHC Class I Restrictd CD8⁺ CTLs Provides AnAssay For In Vivo Tumor Rejection

The effect of vaccination with hsps has been measured thus far in theprior art by tumor rejection assays in vivo. While this assay is clearlythe most demanding and rigorous evidence for immunogenicity, it isimpractical for the purpose of monitoring immune response in humans. Wetested the ability of tumor-derived gp96 preparations to elicit a CD8⁺ Tcell response in order to define an in vitro correlate for in vivo tumorrejection. Mice were immunized twice with 20 micrograms gp96 derivedfrom 6138 or 6139SJ cells. Mixed lymphocyte-tumor cultures (MLTCs)generated from immunized mice were tested in a ⁵¹ Chromium release assayand showed tumor-specific cytotoxicity for the tumor used as the sourceof gp96. This cytotoxic activity could be blocked by anti-MHC class Iantibody K44 (Ozato, K., et al., 1985, Proc. Natl. Acad. Sci. USA82:2427) (FIG. 3A) and by anti-CD8 antibody YTS169.4,(Cobbold, S. P., etal., 1984, Nature 312:548) (not shown). No corresponding activity wasdetected in MLTCs generated from spleens of naive mice. These resultsdemonstrate that vaccination with gp96 elicits effective tumor-specificCTL response, which may be measured in vitro, and independently of thetumor regression responses shown in FIGS. 1 and 2. In light of thegeneral paradigm that exogenous antigens are usually presented throughMHC class II molecules and elicit a helper T cell response (Townsend, A.et al., 1989, Ann. Rev. Immunol. 7:601), the ability of exogenous HSPpreparations to elicit MHC class I-restricted CTLs is unusual.

While testing the ability of tumor-derived gp96 preparations to elicitCTL responses, vaccination with irradiated whole tumor cells was carriedout as a positive control. As expected, vaccination with intactirradiated 6138 cells led to vigorous tumor-specific CTL response.However, vaccination with intact irradiated 6139SJ cells did not lead toa corresponding CTL response (FIG. 3B). This result was surprising asUV-induced cancers of C3H mice are generally highly immunogenic (Kripke,M. L., 1977, Cancer Res. 37:1395. In view of the observation that 6139SJcells are suitable targets for cytotoxic T cells (as seen in FIG. 3A),we deduce that they are not defective in antigen presentation; instead,their inability to elicit CTL response suggests that they. are deficientin a crucial, as yet undefined step necessary specifically for priming aCTL response in vivo. It is most significant in this regard thatalthough 6139SJ cells do not elicit a CTL response, gp96 preparationsderived from them do so efficiently. This suggest that intact-tumorcells and HSPs derived from them elicit immunity through distinctimmunological pathways. The ability of gp96 preparations derived from atumor to elicit a potent CTL response even when the tumor from whichgp96 is derived is unable to do so, makes hsp preparations attractive astherapeutic vaccines.

6.4 GP96-Peptide Complexes Elicit A Memory T Cell Response

The ability to elicit a memory response is crucial for any vaccine andthe ability of gp96 to elicit a memory T cell population was tested. Anumber of criteria, i.e., radiation resistance, kinetics of appearance,loss of CD45RB and L-selectin lymphocyte surface antigens, were used toidentify memory T response. In contrast to naive T cells (Schrek, R.,1961, Ann. N.Y. Acad. Sci 95:839), memory T cells are cycling cells(Mackay, C. R., et al, 1992, Nature 360:264) and like other cyclinglymphocytes, are resistant to sub-lethal irradiation (Lowenthal, J. W.,et al., 1991, Leuc. Biol. 49:388). Thus radiation-resistance can be usedto distinguish naive resting T cells from activated effector and memoryT cells. However, no known surface markers distinguish activatedeffector T cells from memory T cells and the two are distinguishableonly by the kinetics of their appearance. Activated effector T cellsdisappear from circulation within seven to ten days of depletion ofsignificant quantities of antigen (Sprent, J., 1994, Cell 76:315); incontrast, memory T cells continue to circulate well beyond this windowof time. In order to test, if vaccination with tumor-derived gp96elicits a memory T cell response in addition to the effector responseshown in FIG. 3, mice were vaccinated twice at ten day intervals, withtumor-derived gp96 and were irradiated (400 rad) twelve days after thelast vaccination. Three days after irradiation, MLTCs were generatedfrom spleens of mice and tested for tumor-specific CTL response. It wasobserved (FIG. 4) that similar to the response in unirradiated mice(FIG. 3A), the irradiated, gp96-vaccinated mice generated powerful, MHCclass I--restricted and tumor-specific CTL responses. Under this regimenof vaccination and irradiation, the irradiation eliminates thenon-memory resting T cells, while the delay between the last vaccinationand generation of MLTCs eliminates activated T lymphocytes (Sprent, J.,1994, Cell 76:315). Thus, the observed CTL response derives fromradiation-resistant memory T cells elicited by gp96 preparations. Thisphenomenon was also tested in tumor rejection assays in vivo and micevaccinated with pg96 and irradiated were observed to resist-tumorchallenges up to 17 days after vaccination, even though they had beenirradiated (data not shown). These observations indicate thatvaccination with gp96 elicits a long-lived, radiation-resistant T cellpopulation.

As an independent parameter for memory response, expression of CD45RB(Birkeland, H. L., et al., 1989, Proc. Natl. Acad. Sci. USA 86:6734) onCD8⁺ lymphocytes from irradiated and non-irradiated, naive andgp96-vaccinated mice was tested (FIG. 4). In each case, lymphocytes wereobtained under the same regimen as described in the preceding paragraph,i.e., fifteen days after the last vaccination including three days afterirradiation, in order to allow the activated effector cells to bedepleted. It was observed that vaccination with gp96 led to relativeloss of expression of CD45RB on CD8⁺ T lymphocytes in irradiated as wellas non-irradiated, immunized mice. Similar results were observed withL-selectin (data not shown). These results indicated that as judged fromtwo independent sets of criteria, vaccination with gp96 elicits a memoryT cell response. To the best of my knowledge, this is the firstdemonstration of generation of a memory CTL response by vaccination witha biochemically defined, purified cancer vaccine.

7. EXAMPLE ADMINISTRATION OF HSP-PEPTIDE COMPLEXES IN THE TREATMENT OFHEPATOCELLULAR CARCINOMA

Patients with hepatocellular carcinoma are injected with hsp-peptidecomplexes (derived from their own tumors or from other tumors) postsurgery. Treatment with hsp-peptide complexes is started any time aftersurgery. However, if the patient has received chemotherapy, hsp-peptidecomplexes are usually administered after an interval of four weeks ormore so as to allow the immune system to recover. The immunocompetenceof the patient is tested by procedures described in sections 5.7 above.

The therapeutic regimen of hsp-peptide complexes, for example, gp96,hsp90, hsp70 or a combination thereof, includes weekly injections of thehsp-peptide complex, dissolved in saline or other physiologicallycompatible solution.

The dosage used for hsp70 or gp96 is in the range of 10-600 micrograms,with the preferred dosage being 10-100 micrograms. The dosage used forhsp90 is in the range of 50 to 5,000 micrograms, with the preferreddosage being about 100 micrograms.

The route and site of injection is varied each time, for example, thefirst injection is given subcutaneously on the left arm, the secondinjection on the right arm, the third injection on the left abdominalregion, the fourth injection on the right abdominal region, the fifthinjection on the left thigh, the sixth injection on the right thigh,etc. The same site is repeated after a gap of one or more injections. Inaddition, injections are split and each half of the dose is administeredat a different site on the same day.

Overall, the first four to six injections are given at weekly intervals.Subsequently, two injections are given at two-week intervals; followedby a regimen of injections at monthly intervals. The effect ofhsp-peptide complexes therapy is monitored by measuring: a) delayedhypersensitivity as an assessment of cellular immunity; b) activity ofcytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens,e.g., carcinoembryonic (CEA) antigens; d) changes in the morphology oftumors using techniques such as a computed tomographic (CT) scan; and e)changes in putative biomarkers of risk for a particular cancer inindividuals at high risk.

Depending on the results obtained, as described above Section 5.7, thetherapeutic regimen is developed to maintain and/or boost theimmunological responses of the patient, with the ultimate goal ofachieving tumor regression and complete eradication of cancer cells.

8. EXAMPLE ADMINISTRATION OF HSP-PEPTIDE COMPLEXES IN THE TREATMENT OFCOLORECTAL CANCER

Hsp-peptide complexes (gp96, hsp70, hsp90 or a combination thereof) areadministered as adjuvant therapy and as prophylactic adjuvant therapy inpatients after complete reduction of colorectal cancer to eliminateundetectable micrometastases and to improve survival.

The therapeutic and prophylactic regimens used in patients sufferingfrom colorectal cancer are the same as those described in Section 7above for patients recovering with hepatocellular carcinoma. The methodsof monitoring of patients under clinical evaluation for prevention andtreatment of colorectal cancer is done by procedures described inSection 5.7. Specifically, CEA levels are measured as a useful monitorof tumor regression and/or recurrence (Mayer, R. J., et al., 1978,Cancer 42:1428).

9. EXAMPLES METHOD FOR RAPID PURIFICATION OF PEPTIDE-ASSOCIATED HSP70

Hsp70-peptide complexes can be readily obtained from cancer cells orcells infected by an infectious agent or other cells by a rapid,one-step ADP-agarose chromatography, described below.

9.1 Method and Results

Meth A sarcoma cells (500 million cells) were homogenized in hypotonicbuffer and the lysate was centrifuged at 100,000 g for 90 minutes at 4°C. The supernatant was divided into two and was applied to anADP-agarose or an ATP-agarose column. The columns were washed in bufferand were eluted with 3 mM ADP or 3 mM ATP, respectively. The elutedfractions were analyzed by SDS-PAGE: in both cases, apparentlyhomogeneous preparations of hsp70 were obtained. However, when each ofthe preparations was tested for presence of peptides, theADP-bound/eluted hsp70 preparation was found to be associated withpeptides, while the ATP-bound/eluted hsp70 preparation was not. (FIGS.5B and 5A, respectively)

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of eliciting an immune response in ahuman individual of greater than 20 kg body weight comprisingadministering to the individual a composition comprising an amount of apurified complex in the range of 50 to 5,000 micrograms, said complexconsisting essentially of heat shock protein 90 noncovalently bound toan antigenic molecule.
 2. The method according to claim 1 in which theindividual has liver cancer, colon cancer, or breast cancer.
 3. Themethod according to claim 1 in which the amount of the complex is in therange of about 100 micrograms.
 4. The method according to claim 1further comprising administering to the individual an effective amountof a biological response modifier selected from the group consisting ofinterferon-α, interferon-γ, interleukin-2, interleukin-4, interleukin-6,and tumor necrosis factor.
 5. The method according to claim 1 in whichsaid administering step is repeated at weekly intervals.
 6. The methodaccording to claim 1 in which said complex is administeredintramuscularly, subcutaneously, intraperitoneally or intravenously. 7.The method according to claim 1 in which said administering step isrepeated five times, the first administration being on the left arm, thesecond administration being on the right arm, the third administrationbeing on the left belly, the fourth administration being on the rightbelly, the fifth administration being on the left thigh, and the sixthadministration being on the right thigh; said first through sixthadministration being subcutaneously.
 8. A method of treating a humanindividual or greater than 20 kg body weight having a type of cancer,comprising administering to the individual a composition comprising anamount of a purified complex in the range of 50 to 5,000 micrograms,said complex consisting essentially of heat shock protein 90noncovalently bound to an antigenic molecule having an antigenicityspecific to said type of cancer.
 9. The method according to claim 8 inwhich the type of cancer is a sarcoma or carcinoma, selected from thegroup consisting of fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease.
 10. The method according toclaim 8 in which the amount of the complex is 100 micrograms.
 11. Themethod according to claim 8 in which the complex is obtained fromcancerous tissue autologous to the individual.
 12. The method accordingto claim 8 in which the complex is obtained from cancerous tissueallogeneic to the individual.
 13. The method according to claim 8further comprising administering to the individual an effective amountof a biological response modifier selected from the group consisting ofinterferon-α, interferon-γ, interleukin-2, interleukin-4, interleukin-6,and tumor necrosis factor.
 14. The method according to claim 8 in whichsaid administering step is repeated at weekly intervals.
 15. The methodaccording to claim 8 in which said administering step is repeated fivetimes, the first administration being on the left arm, the secondadministration being on the right arm, the third administration being onthe left belly, the fourth administration being on the right belly, thefifth administration being on the left thigh, and the sixthadministration being on the right thigh; said first through sixthadministration being subcutaneously.
 16. The method according to claim 8in which the antigenic molecule is a peptide with which the heat shockprotein is endogenously associated in vivo, and the complex is isolatedfrom cancerous tissue of said type of cancer.
 17. The method accordingto claim 16 in which the cancerous tissue is from the individual. 18.The method according to claim 8 in which the noncovalent complex of theheat shock protein and antigenic molecule is produced in vitro.
 19. Themethod according to claim 18 in which the antigenic molecule is atumor-specific antigen.
 20. A method of inhibiting cancer growth andprogression in a human individual of greater than 20 kg body weight inwhom such inhibition of a type of cancer is desired, comprisingadministering to the individual a composition comprising an amount of apurified complex in the range of 50 to 5,000 micrograms, said complexconsisting essentially of a heat shock protein 90 noncovalently bound toan antigenic molecule having an antigenicity specific to said type ofcancer.
 21. The method according to claim 20, in which the amount of thecomplex is 100 micrograms.
 22. The method according to claim 20 in whichthe antigenic molecule is a peptide with which the heat shock protein isendogenously associated in vivo, and the complex is isolated fromcancerous tissue of said type of cancer.
 23. A method of treating ahuman individual of greater than 20 kg body weight having a type ofcancer, comprising administering to the individual a compositioncomprising an amount of a purified complex in the range of 50 to 5,000micrograms, said complex consisting essentially of heat shock protein 90noncovalently bound to an antigenic molecule, wherein the complex isobtained from tissue of said type of cancer.
 24. The method of claim 23,in which the complex is isolated from cancerous tissue autologous to theindividual.
 25. The method of claim 23, in which the complex is isolatedfrom cancerous tissue allogeneic to the individual.
 26. A method oftreating a human individual of greater than 20 kg body weight having atype of cancer, comprising administering to the individual a compositioncomprising an amount of a purified complex in the range of 50 to 5,000micrograms, said complex consisting essentially of heat shock protein 90noncovalently bound to an antigenic molecule, wherein the complex isobtained from a tumor cell line of said type of cancer.
 27. A method ofinhibiting cancer growth and progression in a human individual ofgreater than 20 kg body weight in whom such inhibition of a type ofcancer is desired, comprising administering to the individual acomposition comprising an amount of a purified complex in the range of50 to 5,000 micrograms, said complex consisting essentially of heatshock protein 90 noncovalently bound to an antigenic molecule, whereinthe complex is obtained from tissue of said type of cancer.
 28. A methodof inhibiting cancer growth and progression in a human individual ofgreater than 20 kg body weight in whom such inhibition of a type ofcancer is desired, comprising administering to the individual acomposition comprising an amount of a purified complex in the range of50 to 5,000 micrograms, said complex consisting essentially of heatshock protein 90 noncovalently bound to an antigenic molecule, whereinthe complex is obtained from a tumor cell line of said type of cancer.29. The method of claim 23, 24, 25, 26, 27, or 28 in which the amount ofthe complex is 100 micrograms.
 30. The method of claim 23, 24, 25, 26,27, or 28 in which the type of cancer is a sarcoma or carcinoma,selected from the group consisting of fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland :arcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease.
 31. The method of claim 30,in which the amounl of the complex is 100 micrograms.
 32. A kitcomprising a container having a composition comprising an amount of apurified complex in the range of 50 to 5,000 micrograms, said complexconsisting essentially of heat shock protein 90 noncovalently bound toan antigenic molecule.
 33. The kit of claim 32 in which the amount ofthe complex is 100 micrograms.